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Molecular diversity analysis of rice (Oryza sativa L.) genotypes using RAPD and SSR marker
Abstract
Random Amplified Polymorphic DNA (RAPD) and Simple Sequence Repeat (SSR) molecular markers were used for detecting the genetic variability of 20 rice genotypes using 79 SSRs and 30 RAPD primers. Among primers used, a set of 16 SSR and 14 RAPD markers showed polymorphism, and banding patterns were scored as 1 (present) or 0 (absent) in the datasheet which was further analyzed by using Jaccard’s similarity coefficient and SAHN clustering. The number of alleles, PIC value, and heterozygosity for individual 16 SSR and 14 RAPD markers were used to assess the degree of polymorphism among the rice genotypes. A total of 36 polymorphic loci were found in 16 polymorphic SSR markers and 77 polymorphic loci were observed in 14 polymorphic RAPD markers. The number of alleles produced per locus ranged from 2 (RM447, RM21, RM171, RM237, RM25, RM283, RM510, RM259, RM334, RM433, RM489, and RM212) to 3 (RM263, RM152, RM413, and RM3331) in 16 SSRs markers and 3 (OPR-05) to 9 (OPR-02 and OPC-06) in 14 RAPD. In SSRs, the PIC value diverse from 0.30 (RM447) to 0.58 (RM152) with an average of 0.38 per locus and an average value of heterozygosity (0.49). RAPD analysis showed an average PIC value of 0.78. Based on the information generated, the 20 rice genotypes were grouped into two main clusters in both the analysis of the marker. In SSR markers analysis, cluster I comprised 4 genotypes and cluster II comprised 16 genotypes and in RAPD cluster I was shown 2 genotypes and cluster II show 18 genotypes.
... Expression of morphological characters is governed by many genes; therefore, remarkably controlled by ecological factors and crop stages of development. Thus, analysis of genetic diversity based on those morphological traits is treated as inefficient while DNA or molecular markers are at the top of polymorphism and free from environmental connections [6,7]. These genetic markers have been utilized efficiently and are reported as the best technique to understand genetic range and relationship [8,9]. ...
The present investigation was carried out for the assessment of molecular diversity in parental lines of muskmelon to be used for the crossing programs to get hybrids with desirable traits. A total of eight inbred lines of andromonoecious muskmelon were assessed with twenty ISSR (Inter Simple Sequence Repeat) primers. Based on polymorphism four primers were selected for whole molecular analysis. The polymorphism varied from 50.00 to 90.90% with an average of 82.97 percent. Pair-wise similarity matrix in accordance with Jaccard’s coefficient revealed the similarity estimation ranging from 55.00% to 75.00%. The maximum similarity value was recorded for the PMM-37 (0.75) with Hara Madhu while the lowest was observed for PMM-4A (0.55) with PMM-16 and between PMM-32 (0.55) and PMM-18. Eight genotypes were categorized into two main clusters. Cluster I included 6 genotypes and Cluster II comprised 2 genotypes with approximately 70.80% similarity among themselves. Cluster I is further subdivided into two sub-clusters IA and IB with 63.00% similarity. Sub-cluster IA further forked into two small clusters IAi and IAii. IAi is comprised of PMM 4A. IAii is further subdivided into 2 groups; Group 1 and Group 2 with approximately 73.60% similarity. Group 1 consists of PMM 16 and group 2 consists of PMM 37 and check variety Hara Madhu with approximately 74.00% similarity. Sub-cluster IB comprised two genotypes PMM 32 and PMM 18 with approximately 70.90% similarity among themselves.
Studies on genetic diversity and relationships are necessary for breeders and scientists to increase the effectiveness of future breeding programs. The tidal swamp rice (Oryza sativa L.) is one of the potential germplasms which has a prominent opportunity to be incorporated in the rice breeding program. This study aimed to investigate and reveal the genetic diversity and relationship of tidal swamp rice germplasms indigenously from South Kalimantan, Indonesia, using Random Amplified Polymorphic DNA (RAPD) markers. A total of ten rice samples, consisting of nine from this region and one from South Sumatera (an outgroup), and five selected RAPD markers, i.e., OPB-06, OPAJ-01, OPAB-17, OPAL-09, and OPAL-08, were used in this study. DNA amplifications were performed and programmed for one cycle of initial denaturation (5 min, 94ºC), 45 cycles of denaturation (30 sec, 94ºC), annealing (30 sec, 37ºC), and extension (1.5 min, 72ºC), as well as one cycle of final extension (7 min, 72ºC). The genetic similarity was analyzed using Dice’s coefficient method, whereas their relationship (dendrogram) by the UPGMA. The results showed that these germplasms have a moderate genetic diversity level, indicated by the polymorphism degree of 75.64%. The clustering analysis revealed that they are grouped into three main groups at a similarity coefficient of 0.70. In this case, Siam Unus is distantly related to the other cultivars and forms a solitaire group. Siam Unus also shows the farthest relationship with Sardani, an outgroup. It is a new finding for the genetic insight of tidal swamp rice of South Kalimantan, Indonesia, including their diversity and relationship. Thus, the results obtained from this study is useful in supporting future rice conservation and breeding programs.
Background
Knowledge of the genetic diversity and population structure of germplasm collections is an important foundation for crop improvement. Rice production across a broad range of rice-growing environments results in a diverse array of local rice varieties. Many rice varieties have been lost as a result of biodiversity loss and are now grown in Pakistan.Methods and resultsTo protect the biodiversity of rice varieties, an experiment was carried out to check the genetic and morphological variations between 8 exotic and 7 local rice genotypes, using 5 different SSR markers, i.e., RM3, RM259, RM341, RM520, and RM11943. The analysis of morphological and quality traits of rice observed significant variation across genotypes. The results revealed that genotype Irri-Pak attained the highest plant height and primary branch plant-1, while genotype Mushkan produced a higher number of productive tillers and obtained a higher fertility factor (%). Similarly, the highest value for panicle length was observed for genotype Faker-e-Malakand, 1000-grains weight in genotype Calmochi, and maximum days to maturity was noticed in genotype Swati-2014. Moreover, the genotype Brio attained the highest value of stem diameter, while maximum seed length was noted in the genotype Sug Dasi. The highest number of primary branches plant−1 in genotype Ibge-I and secondary branches plant−1 in genotype Calmochi were noticed. A higher concentration of sodium and potassium was observed for the genotype Marte, while the genotype Muskan attained the maximum content of copper. Moreover, the highest concentration of iron in genotype Originario, zinc in genotype JP-5, and cadmium content were noticed in genotype Ibge. Similarly, the dendrogram analysis for quantitative parameters showed three clusters at 74.13% similarities. Whereas all the genotypes of European origin formed a separate cluster. A set of 5 simple sequence repeat primers, covering four chromosomes, amplified a total of 14 alleles and showed 100% polymorphism with an average PIC value ranging from 0.39 to 0.91. The UPGMA cluster analysis separated the 15 rice genotypes into 3 main groups based on 32.5% similarities and the highest genetic distance (45.1%) was observed between two genotypes (Fakher-e-malakand and Musa), having different geographical origins. There was no genetic distance between the genotypes Marte and Brio, irrespective of having the same origin.Conclusions
The maximum genetic distances were noted for genotype, Fakhre-e-Malakand and Musa having a different origin, while the minimum genetic distance was shown by genotypes, Marte and Onice, from the same origin.
The present investigation was carried to know the extent of genetic variability in sixty-five germplasm lines of rice collected from all over India, for yield and yield attributing characters. Analysis of variance revealed highly significant differences for all the 10 characters, indicating the presence of genetic variability among the genotypes. The present investigation revealed that the estimates of PCV were slightly higher than GCV for all the characters studied indicating less influence of environmental factors on the expression of traits and the possibility for genetic improvement through direct selection for these traits. The magnitude of PCV and GCV estimates were moderate for three traits viz., days to 50% flowering, plant height, panicle length, total number of tillers per plant, effective number of tillers per plant and test weight, indicating the influence of environment rather than the genotype alone. The heritability and genetic advance estimates were moderate for panicle length and high for all the other traits viz., days to 50% flowering, plant height, total number of tillers per plant, effective number of tillers per plant, panicle weight, number of filled grains per panicle, number of unfilled grains per panicle, test weight and single plant yield indicating the influence of additive gene action, as such selection would likely be effective for improvement of these traits.
Rice is critical for sustainable food and nutritional security; however, nominal micronutrient quantities in grains aggravate malnutrition in rice-eating poor populations. In this study, we evaluated genetic diversity in grain iron (Fe) and zinc (Zn) contents using trait-linked simple sequence repeat (SSR) markers in the representative subset of a large collection of local and exotic rice germplasm. Results demonstrated that aromatic fine grain accessions contained relatively higher Fe and Zn contents in brown rice (BR) than coarse grain accessions and a strong positive correlation between both mineral elements. Genotyping with 24 trait-linked SSR markers identified 21 polymorphic markers, among which 17 demonstrated higher gene diversity and polymorphism information content (PIC) values, strongly indicating that markers used in current research were moderate to highly informative for evaluating the genetic diversity. Population structure, principal coordinate and phylogenetic analyses classified studied rice accessions into two fine grain specific and one fine and coarse grain admixture subpopulations. Single marker analysis recognized four ZnBR and single FeBR significant marker-trait associations (MTAs) contributing 15.41–39.72% in total observed phenotypic variance. Furthermore, high grain Fe and Zn contents linked marker alleles from significant MTAs were also identified. Collectively, these results indicate a wide genetic diversity exist in grain Fe and Zn contents of studied rice accessions and reveal perspective for marker-assisted biofortification breeding.
A set of 24 of SSR markers were used to estimate the genetic diversity in 16 rice genotypes found in Western Himalayas of Kashmir and Himachal Pradesh, India. The level of polymorphism among the genotypes of rice was evaluated from the number of alleles and PIC value for each of the 24 SSR loci. A total of 68 alleles were detected across the 16 genotypes through the use of these 24 SSR markers The number of alleles per locus generated varied from 2 (RM 338, RM 452, RM 171) to 6 (RM 585, RM 249, RM 481, RM 162). The PIC values varied from 0.36 (RM 1) to 0.86 (RM 249) with an average of 0.62 per locus. Based on information generated, the genotypes got separated in six different clusters. Cluster 1 comprised of 4 genotypes viz; Zag 1, Zag 13, Pusa sugandh 3, and Zag 14, separated from each other at a similarity value of 0.40. Cluster second comprised of 3 landraces viz; Zag 2. Zag 4 and Zag10 separated from each other at a similarity value of 0.45. Cluster third comprised of 3 genotypes viz; Grey rice, Mushk budji and Kamad separated from each other at a similarity value of 0.46. Cluster fourth had 2 landraces viz; Kawa kreed and Loual anzul, and was not sub clustered. Fifth cluster had 3 genotypes viz; Zag 12, Purple rice and Jhelum separated from each other at a similarity value of 0.28. Cluster 6 comprised of a single popular variety i.e. Shalimar rice 1 with independent lineage.
Random Amplified Polymorphic DNA (RAPD) analysis was performed to assess the genetic diversity in ten selected cultivars of rice, Oryza sativa L. using 30 decamer random primers. Out of 30, 25 RAPD primers revealed polymorphism while the remaining 5 primers showed no reaction. The primers produced a total of 428 bands of which 363 were polymorphic (85.02%). The number of polymorphic fragments for each primer varied from 7 to 23 with an average of 14 polymorphic fragments. The primer OPB-17 produced the maximum number of polymorphic bands. The RAPD data was analyzed to determine the genetic similarity coefficients which ranged from 0.46 to 0.81. Cluster analysis was performed using Unweighted Paired Group of Arithmetic Means (UPGMA) using the Jaccard's similarity coefficient. The UPGMA dendrogram resolved the selected rice cultivars into two major clusters.
Rice is one of the most important basic foods, especially in developing countries. However, rice production is negatively affected by drought and decreased water availability. Over the recent decades, aerobic rice has been considered as a promising approach to reduce water input. However, the rice genotypes suitable for aerobic rice should also be evaluated for other important characteristics such as grain micronutrient content. Studying the genetic variability of grain Fe, Mn, Zn, and protein content in aerobic rice genotypes compared to other rice varieties can be useful for improving nutritive value via plant breeding programs. In this study, 50 rice genotypes, including aerobic rice, Iranian-improved, and landrace varieties were studied for Fe, Mn, Zn, and protein content in brown rice and also for their association with 77 SSR markers. There was a significant diversity among the rice genotypes: 13.91–44.91 mg/kg for Fe, 5.85–35.93 mg/kg for Mn, 11.79–34.33 mg/kg for Zn, and 4.38–16.63% for protein. According to the cluster analysis, group (I) contained five Iranian landrace rice varieties (Champaboudar, Gharib, Alikazemi, Anbarbou, and Sangetarom) well-known for their eating and cooking quality and aroma; furthermore, 10 aerobic rice genotypes had the highest level of Fe and Zn content among other groups. According to the results some aerobic rice genotypes had high grain micronutrient values such as IR 82,639-B-B-103–4, which also had 16.63% protein content in the grain. Based on the results of association analysis, out of 55 random SSRs, 11 markers, and out of 22 linked SSRs, 17 markers were found to be associated with grain protein, Fe, Zn, and Mn in the present population. Out of 28 significant markers, RM488 associated with grain Fe, RM574 with Mn, and RM248 with Mn and Zn and the associated was detected in both the years. In addition, out of 11 newly detected SSR markers, five were substantially associated with rice grain quality traits. The markers, which are showing association, can be utilized to enhance the nutritional value of rice in plant breeding programs.
Genetic improvement mainly depends on the extent of genetic variability present in the population. The molecular marker is a useful tool for assessing genetic variations and resolving cultivar identities. The objective of this study was to evaluate the genetic divergence of 30 rice varieties (Basmati, Non-Basmati, Aerobic) using 10 ISSR, RAPD markers each. The diversity or similarities and dissimilarities between all thirty rice varieties were calculated using 0 1 sheets. SSR primers RM-263 is highly informative since it recorded high PIC value (0.995). The resolving power varies between 0.132(RM-256) to 4.662(RM-222) with an average value of 2.7502. In RAPD analysis PIC values varies from 0.811(OPD-08) to 0.9925(OPF-13) with average of 0.9635 and resolving power varies from 1.32(OPJ-08) to 2.066(OPJ-13) with average of 1.8256. In ISSR analysis, PIC value ranged from 0.8791(ISSR6) to 0.9916(ISSR5)
with an average value of 0.9482. The resolving power varies between 1.6(ISSR3) and 8.366(ISSR2) with an average value of 5.2708. The PIC values and Resolving power were calculated for individual primers. The analysis indicated that ISSR expressed maximum resolving power of 8.336 and RAPD gave maximum PIC values of 0.9925. RAPD primer OPF-13 gave the maximum accessions coverage (depending on the value of PIC) in the rice genome. Out of 52 amplified bands, 49 bands were polymorphic and 3 bands were monomorphic. The cluster analysis using the marker systems could distinguish the different genotypes. The dendogram generated on the principle of Unweight Pair Wise Method using Arithemetic Average (UPGMA) was constructed by Jaccard’s Coefficient and the genotypes were grouped in to clusters. The dendogram developed for aroma and quality traits showed that the genotypes with common phylogeny and geographical orientation tend to cluster together thus marker based molecular fingerprinting could serve as a sound basis in the identification of genetically distant accessions as well as in the duplicate sorting of the morphologically close accessions as the case is common in differentiating Basmati and non-basmati.
Germplasm diversity is the mainstay for crop improvement and genetic dissection of complex traits. Understanding genetic diversity, population structure, and the level and distribution of linkage disequilibrium (LD) in target populations is of great importance and a prerequisite for association mapping. In this study, 100 genome-wide simple sequence repeat (SSR) markers were used to assess genetic diversity, population structure, and LD of 416 rice accessions including landraces, cultivars and breeding lines collected mostly in China. A model-based population structure analysis divided the rice materials into seven subpopulations. 63% of the SSR pairs in these accessions were in LD, which was mostly due to an overall population structure, since the number of locus pairs in LD was reduced sharply within each subpopulation, with the SSR pairs in LD ranging from 5.9 to 22.9%. Among those SSR pairs showing significant LD, the intrachromosomal LD had an average of 25-50 cM in different subpopulations. Analysis of the phenotypic diversity of 25 traits showed that the population structure accounted for an average of 22.4% of phenotypic variation. An example association mapping for starch quality traits using both the candidate gene mapping and genome-wide mapping strategies based on the estimated population structure was conducted. Candidate gene mapping confirmed that the Wx and starch synthase IIa (SSIIa) genes could be identified as strongly associated with apparent amylose content (AAC) and pasting temperature (PT), respectively. More importantly, we revealed that the Wx gene was also strongly associated with PT. In addition to the major genes, we found five and seven SSRs were associated with AAC and PT, respectively, some of which have not been detected in previous linkage mapping studies. The results suggested that the population may be useful for the genome-wide marker-trait association mapping. This new association population has the potential to identify quantitative trait loci (QTL) with small effects, which will aid in dissecting complex traits and in exploiting the rich diversity present in rice germplasm.
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